System and method for forming bioengineered tubular graft prostheses

ABSTRACT

An apparatus for forming a tube construct from a planar sheet matrix includes a stand supporting two opposing mounts and spanning between the opposing mounts are a mandrel, a porous rod, and a spring-loaded roller held in parallel arrangement; a guide for aligning and engaging the mandrel on the opposing mounts; and a means for imparting a tangential force on the planar sheet matrix to prevent wrinkling. The porous rod has a lumen running its length and has pores that communicate between the lumen of the porous rod through to the surface of the rod for water to uniformly pass through. The spring-loaded roller runs along the length of the porous rod creating a line of contact between the roller and the mandrel. The mandrel is contacted with a planar sheet of matrix and is rotated such that successive portions of the matrix contact the porous rod and become lightly moistened by the water passing through the pores of the porous rod and become wrapped around the mandrel to form a tube construct.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional Application of U.S. patent applicationSer. No. 10/325,444, filed Dec. 19, 2002, which claims the benefit ofU.S. Provisional Application Ser. No. 60/342,831 entitled “SYSTEM ANDMETHOD FOR FORMING BIOENGINEERED TUBULAR GRAFT PROSTHESES” filed on Dec.21, 2001, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention is in the field of tissue engineering. The invention isdirected to a system and a method for preparing bioengineered graftprostheses prepared from cleaned tissue material derived from animalsources. The bioengineered graft prostheses made using the invention aretubular, of small diameter, and have a uniform geometry along theirentire length. The bioengineered graft prostheses are used forimplantation, repair, or for use in a mammalian host.

BACKGROUND OF THE INVENTION

The present invention overcomes the difficulties in forming a fine gaugetube of uniform geometry from processed tissue matrix or reconstitutedmatrix.

SUMMARY OF THE INVENTION

The invention is a system for fabricating tubular constructs from planarsheet-like processed tissue matrices or reconstituted matrices. Thesystem comprises two devices: a flagging device and a rolling device.Each device accommodates a mandrel on which the tubular construct isformed. First, a matrix is flagged on the mandrel using the flaggingdevice. Second, the matrix is then rolled onto the mandrel using therolling device.

Therefore, the method of the invention comprises: (a) a method forflagging a sheet of processed tissue matrix by aligning a mandrel alongone edge of the sheet and contacting it to the sheet so that the sheetand the matrix adhere, and (b) rolling the flagged sheet around themandrel while maintaining even tension on the sheet and smoothing outbubbles or creases as it is rolled onto the mandrel. Rolling continuesuntil the sheet contacts and overlaps itself to a degree. The overlap isthe bonding region that keeps the tissue in a tubular form.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a view of the flagging apparatus of the invention.

FIG. 2 shows a side cross-sectional view of the rolling apparatus of theinvention.

FIG. 3 shows a three-dimensional view of the rolling apparatus of theinvention.

DETAILED DESCRIPTION

The invention is directed toward a system and methods for makingtubular-shaped tissue engineered prostheses from thin planar materialswhere the system and methods do not require adhesives, sutures, orstaples to bond the tissue in a tubular form and consequently maintainthe bioremodelability of the prostheses.

Advantages provided by the invention are that the apparatus can makeconstructs faster and more consistently than if made manually. Thesystem devices of the invention aid in even circumferential tensioningand radial compression of the tissue which smoothes out air or waterbubbles or creases that can occur under the mandrel or between thelayers of the tube. Because the constructs are used as medical devices,minimal variations can potentially affect the functional performance ofthe constructs when implanted in a patient.

The terms “processed tissue matrix” and “processed tissue material” meannative, normally cellular tissue that has been procured from an animalsource, preferably a mammal, and mechanically cleaned of attendanttissues and chemically cleaned of cells, cellular debris, and renderedsubstantially free of non-collagenous extracellular matrix components.The processed tissue matrix, while substantially free cellular debris,maintains much of its native matrix structure, strength, and shape.Preferred compositions for preparing the bioengineered grafts of theinvention are animal tissues comprising collagen, including, but notlimited to: intestine, fascia lata, pericardium, dura mater, and otherflat or planar structured tissues that comprise a fibrous tissue matrix.The planar structure of these tissue matrices makes them able to beeasily manipulated and assembled using the devices and methods of theinvention. A more preferred composition for preparing the bioengineeredgrafts of the invention is an intestinal collagen layer derived from thetunica submucosa of small intestine. Suitable sources for smallintestine are mammalian organisms such as human, cow, pig, sheep, dog,goat, or horse while small intestine of pig is the preferred source. Themost preferred composition for preparing tubular prostheses using theinvention is a processed intestinal collagen layer derived from thetunica submucosa of porcine small intestine. To obtain the processedintestinal collagen layer, the small intestine of a pig is harvested andattendant mesenteric tissues are grossly dissected from the intestine.The tunica submucosa is preferably separated, or delaminated, from theother layers of the small intestine by mechanically squeezing the rawintestinal material between opposing rollers to remove the muscularlayers (tunica muscularis) and the mucosa (tunica mucosa). The tunicasubmucosa of the small intestine is tougher than the surrounding tissue,hence the rollers squeeze the more friable components from thesubmucosa. In the examples that follow, the tunica submucosa wasmechanically harvested from porcine small intestine using a Bitterlinggut cleaning machine and then chemically cleaned to yield a cleanedtissue matrix as described in U.S. Pat. No. 5,993,844, the disclosure ofwhich is incorporated herein by reference. This mechanically andchemically cleaned intestinal collagen layer is herein referred to as“ICL”. ICL is used to prepare tubular constructs that are used asbioengineered medical devices such as those described in InternationalPCT Application Publication Nos. WO 95/22301, WO 99/62424, WO 99/62425,and WO 99/62427, the teachings of which are incorporated herein byreference.

The terms, “reconstituted matrix” and “reconstituted material”, meananimal-derived or cell-derived matrix components that have beenextracted and purified from either tissues or cell cultures. The matrixmay be formed from solubilized matrix components, principally collagensuch that the matrix has tissue-like properties with regard to structureand physical properties. The reconstituted matrix may be highly purifiedand may have other components added to the matrix when the matrix isreformed. Other suitable collagenous tissue sources or other nativetissue, reconstituted matrix sheets, or synthetic materials with thesame flat sheet structure may be identified by the skilled artisan inother animal sources.

In the description of the devices and methods of the invention, and inthe examples that follow, a sheet-like material, preferably either aprocessed tissue matrix or a reconstituted matrix, is used to make thetubular constructs. While not intending to be so limited but forsimplicity in illustration of the apparatus and methods of theinvention, and to describe the most preferred embodiment, thefabrication of a tube from a sheet of ICL will be described.

In the first aspect of the system of the invention, a flagging device isemployed. Flagging introduces the ICL to be tubulated to a mandrel onwhich the tubular construct is formed. Referring to FIG. 1, shown is theflagging device of the invention. The flagging device 10 comprises abase platform 12 with legs 14. The platform incorporates a hollow chuck16 with a plurality of machined holes 18 on its top facing surface, thatcommunicate between the inside and outside of the chuck, and a port 20.The port 20 is connected to a vacuum source. Running along the surfaceof the platform 12 and along one edge of the hollow chuck 16 is a groove22. The groove 22 accommodates a cylindrical mandrel 24 that is coveredwith an elastic sleeve and supported at each end by mandrel holders 25.The starting material, either a processed tissue matrix, such as ICL, ora reconstituted matrix, has a sheet-like geometry, preferably with atleast one straight edge, more preferably rectangular. The ICL is driedin air before use. The sleeve on the mandrel is wetted with sterilewater. The ICL is placed on the top surface of the platform with oneedge of the material aligned along the center of the mandrel. The vacuumsource is turned on to pull air through the machined holes 18 in the topof the hollow chuck 16. Because the vacuum is on, the ICL is held flatand even against the platform. The material is then contacted to thesleeve on the mandrel by raising the mandrel holders 25 so that only oneedge of the ICL is contacted to the mandrel and is moistened by thewater on the sleeve. The ICL, sticky when moistened, adheres to themandrel. The ICL is allowed to dry to a point where it will remainadhered to the mandrel when the mandrel is lifted from the groove in theplatform. A rectangular piece of ICL, when adhered to the mandrel alongone edge, will resemble a flag.

The second aspect of the system of the invention is a device for forminga tube from flagged ICL. Referring to FIG. 2, shown is the rollingdevice of the invention. The rolling device 50 comprises a stand 51 thatsupports two opposing mounts 52. Passing between and held in parallelarrangement by the opposing mounts are a porous tubular ceramic rod 55,a hollow chuck 57, and a spring-loaded roller 60. The ceramic rod 55 hasa lumen running its length with one end of the ceramic rod is closed andthe other end extending beyond the mount and open to serve as a port.Pores communicate between the lumen of the rod through to the surfacefor water to uniformly pass through. Above the level of the ceramic roda hollow chuck 57 with machined holes that communicate between theinterior and exterior of the chuck. The hollow chuck has a plurality ofholes on the face towards the roller 60 and a port at one end for theattachment of a vacuum source. The spring-loaded roller 60 runs alongthe length of the ceramic rod creating a line of contact between theroller 60 and the ICL on mandrel 24. In each mount, the spring-loadedroller 60 is contacted by an end of a perpendicular rod 62 loaded by acoil spring contained in the mount. The perpendicular rod 62 passesthrough the mount via an extender rod 67. The perpendicular rods 62 canbe disengaged from the roller 60 by engaging a solid bar 68 between theends of the extender rods 67 and the spring housings 64. In each of theopposing mounts is a guide member 70 having an L-shaped groove where thetop of the guide is open to accommodate one end of the mandrel and thebottom of the guide aligns the mandrel to engage it against the ceramicrod. When the spring-loaded roller is disengaged, the guides are openfor the insertion of a mandrel between the opposing mounts. When theguides are loaded with a mandrel and the spring-loaded roller isengaged, the roller presses against the mandrel on one side such thatthe mandrel contacts the ceramic rod on the opposite side.

Before loading the guide with a mandrel, the vacuum and water sourcesare activated so that air is pulled through the machined holes in thehollow chuck to the interior of the chuck and the water is slowlypassing from the lumen of the ceramic tube to its surface. The ends ofthe mandrel with the flagged ICL are placed in the guides with the freeend of the flagged ICL upright and away from the rolling device. Thespring-loaded roller is actuated against the mandrel forcing the mandrelto contact the porous ceramic rod. The mandrel is then rotated to wrapthe ICL around the mandrel. The ICL is held taught by the vacuum fromthe hollow chuck 57. As the mandrel is rotated, successive portions ofthe ICL contact the porous ceramic rod and are lightly moistened by thewater flowing out of the ceramic rod. The mandrel is rotated until theentire piece of ICL is wrapped around the mandrel.

The bioengineered constructs produced by the devices and methods of theinvention are tubular in shape and may be formed to any length orthickness. The length of the construct is limited only by the size ofthe devices of the system and the length of the mandrel and the longestdimension of a sheet of material. The thickness of the construct may bechosen so that the final construct is one or more layers, depending onthe number of times the mandrel that holds the sheet of material isrotated, with the limitation being the maximum thickness that theapparatus can manage. For a single layer construct, there will be someoverlap where a bonding region is formed to maintain the tubular shapeof the final construct. The diameter of the tube is determined by thediameter of the mandrel chosen.

To form a tubular construct, a mandrel is chosen with a diametermeasurement that will determine the final inner diameter of the formedtube construct. The mandrel is preferably cylindrical or oval in crosssection and made of glass, stainless steel, ceramic, or plastic andpreferably of a nonreactive, medical grade composition. The number oflayers intended for the tubular construct to be formed corresponds withthe number of times an ICL is wrapped around a mandrel and over itself.The number of times the ICL can be wrapped depends on the width of theprocessed ICL sheet. For a two layer tubular construct, the width of thesheet must be sufficient for wrapping the sheet around the mandrel atleast twice. Similarly, the length of the mandrel will dictate thelength of the tube that can be formed on it. For ease in handling theconstruct on the mandrel, the mandrel should be longer than the lengthof the construct so the mandrel, and not the construct being formed, iscontacted when handled.

It is preferred that the mandrel is provided with an elastic sleeve. Thesleeve may be a nonreactive, medical grade quality, elastomericmaterial. While a tubular ICL construct may be formed directly on themandrel surface, the sleeve facilitates the removal of the formed tubefrom the mandrel and does not adhere to, react with, or leave residueson the ICL. To remove the formed construct, the sleeve may be pulled offfrom one end of the mandrel and carry the construct from the mandrelwith it. Because the processed ICL only lightly adheres to the sleeveand is more adherent to other ICL layers, fabricating ICL tubes isfacilitated as the tubulated construct may be removed from the mandrelwithout stretching or risking damage to the tube construct. In the mostpreferred embodiment, the elastic sleeve comprises KRATON® (ShellChemical Company), a thermoplastic rubber composed ofstyrene-ethylene/butylene-styrene copolymers with a very stablesaturated midblock.

For illustration, a two-layer tubular construct with a 4 mm innerdiameter and an additional 20% overlap is formed on a mandrel havingabout a 4 mm diameter. The mandrel is provided with a KRATON® sleeveapproximately as long as the length of the mandrel and longer than theconstruct to be formed on it. A sheet of ICL is trimmed so that thewidth dimension is about 28 mm and the length dimension may varydepending on the desired length of the construct. In the sterile fieldof a laminar flow cabinet, the ICL is then formed into an ICL collagentube by the following process. The ICL is moistened along one edge andis aligned with the sleeve-covered mandrel and, leveraging the adhesivenature of the ICL, it is “flagged” along the length of thesleeve-covered mandrel and dried in position for at least 10 minutes.The flagged ICL is then hydrated and wrapped around the mandrel and thenover itself one full revolution plus 20% of the circumference, for a120% total overlap, to serve as a bonding region and to provide a tightseam. To obtain a tubular construct with the mucosal side of the ICL asthe lumen of the formed construct, the mucosal side of the ICL ismoistened along one edge, flagged on the mandrel, and wrapped so thatthe mucosal side of the ICL faces the mandrel. Using the method above, atubular construct can be made with the mucosal side of the ICL as thelumen or, alternatively, the serosal side of the ICL as the lumen byorienting the ICL appropriately during flagging.

For the formation of single layer tubular construct, the ICL must beable to wrap around the mandrel one full revolution and at least about a5% additional revolution as an overlap to provide a bonding region thatis equal to about 5% of the circumference of the construct. For atwo-layer construct, the ICL must be able to wrap around the mandrel atleast twice and preferably an additional 5% to 20% revolution as anoverlap. While the two-layer wrap provides a bonding region of 100%between the ICL surfaces, the additional percentage for overlap ensuresa minimum of 2 layers throughout the graft. For a three-layer construct,the ICL must be able to wrap around the mandrel at least three times andpreferably an additional 5% to 20% revolution as an overlap. Theconstruct may be prepared with any number of layers depending on thespecifications for a graft required by the intended indication.Typically, a tubular construct will have 10 layers or less, preferablybetween 2 to 6 layers and more preferably 2 or 3 layers with varyingdegrees of overlap. During and after wrapping, any air bubbles, folds,and creases are smoothed out from under the material and between thelayers.

The layers of the wrapped ICL are then bonded together by dehydratingthem while in wrapped arrangement on the sleeve-covered mandrel. Whilenot wishing to be bound by theory, dehydration brings the extracellularmatrix components, such as collagen fibers, in the layers together whenwater is removed from the spaces between the fibers in the matrix.Dehydration may be performed in air, in a vacuum, or by chemical meanssuch as by acetone or an alcohol such as ethyl alcohol or isopropylalcohol. Dehydration may be done to room humidity, normally betweenabout 10% RH to about 50% RH. Dehydration may be performed by placingthe mandrel with the ICL layers into the oncoming airflow of a laminarflow cabinet for at least about 1 hour up to 24 hours at ambient roomtemperature, approximately 20° C., and at room humidity. At this pointthe wrapped dehydrated ICL constructs may be then pulled off the mandrelvia the sleeve or left on for further processing. The constructs may berehydrated in an aqueous solution, preferably water, by transferringthem to a room temperature container containing rehydration agent for atleast about 10 to about 15 minutes to rehydrate the layers withoutseparating or delaminating them. The thus formed collagen tube constructis then used to form a prosthesis, preferably a bioremodelableprosthesis.

The constructs are then preferably crosslinked together by contactingthem with a crosslinking agent, preferably a chemical crosslinking agentthat preserves the bioremodelability of the ICL material. As mentionedabove, the dehydration brings the extracellular matrix components ofadjacent ICL layers together for crosslinking those layers of the wraptogether to form chemical bonds between the components and thus bond thelayers together. Alternatively, the constructs may be rehydrated beforecrosslinking by contacting an aqueous solution, preferably water, bytransferring them to a room temperature container containing rehydrationagent for at least about 10 to about 15 minutes to rehydrate the layerswithout separating or delaminating them. Crosslinking the bondedprosthetic device also provides strength and durability to the device toimprove handling properties. Various types of crosslinking agents areknown in the art and can be used such as ribose and other sugars,oxidative agents and aldehydes. A preferred crosslinking agent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Inan another preferred method, sulfo-N-hydroxysuccinimide is added to theEDC crosslinking agent as described by Staros, J. V., Biochem, 21,3950-3955, 1982. Besides chemical crosslinking agents, the layers may bebonded together by physical means such as dehydrothermal (DHT) andultraviolet (UV) methods or by other methods such as with fibrin-basedglues or medical grade adhesives including cyanoacrylate, polyurethane,vinyl acetate or polyepoxy. In the most preferred method, EDC issolubilized in water at a concentration preferably between about 0.01 mMto about 100 mM, more preferably between about 0.1 mM to about 10 mM,most preferably at about 1.0 mM. Besides water, phosphate bufferedsaline or (2-[N-morpholino]ethanesulfonic acid) (MES) buffer may be usedto dissolve the EDC. In addition, other agents may be added to thesolution such as acetone or an alcohol may be added up to 99% v/v inwater to modulate the crosslinking. EDC crosslinking solution isprepared immediately before use as EDC will lose its activity over time.To contact the crosslinking agent to the ICL, the hydrated, ICL tubularconstructs are transferred to a container such as a shallow pan and thecrosslinking agent gently decanted to the pan ensuring that the ICLlayers are both covered and free-floating and that no air bubbles arepresent under or within the layers of ICL constructs. The pan is coveredand the layers of ICL are treated with crosslinking agent for betweenabout 4 to about 24 hours after which time the crosslinking solution isdecanted and disposed of.

Constructs are rinsed in the pan by contacting them with a rinse agentto remove residual crosslinking agent. A preferred rinse agent is wateror other aqueous solution. Preferably, sufficient rinsing is achieved bycontacting the chemically bonded constructs three times with equalvolumes of sterile water for about five minutes for each rinse. If theconstructs have not been removed from the mandrels, they may be removedat this point by pulling the sleeves from the mandrels. The constructsare then allowed to dry and when dry, the sleeve may be removed from thelumen of the constructs simply by pulling it out by one of the freeends.

In embodiments where the construct will be used as a vascular graft, theluminal surface of the construct may be rendered less thrombogenic byapplying a deposited collagen layer or heparin, or both, to the lumen ofthe formed tube. Heparin can be applied to the prosthesis by a varietyof well-known techniques. For illustration, heparin can be applied tothe prosthesis in the following three ways. First, benzalkonium heparin(BA-Hep) isopropyl alcohol solution is applied to the prosthesis byvertically filling the lumen or dipping the prosthesis in the solutionand then air-drying it. This procedure treats the collagen with anionically bound BA-Hep complex. Second, EDC can be used to activate theheparin and then to covalently bond the heparin to the collagen fiber.Third, EDC can be used to activate the collagen, then covalently bondprotamine to the collagen and then ionically bond heparin to theprotamine. Many other coating, bonding, and attachment procedures arewell known in the art that could also be used.

The following examples are provided to better elucidate the practice ofthe present invention and should not be interpreted in any way to limitthe scope of the present invention. Those skilled in the art willrecognize that various modifications can be made to the methodsdescribed herein while not departing from the spirit and scope of thepresent invention.

EXAMPLES Example 1 Method for Making an ICL Tube Construct

In the sterile field of a laminar flow cabinet, the ICL was formed intoICL collagen tubes by the following process. Lymphatic tags were trimmedfrom the serosal surface of the ICL. The ICL was blotted with sterileabsorbent towelettes to absorb excess water from the material and thenspread on a porous polycarbonate sheet and dried in the oncoming airflowof the laminar flow cabinet. Once dry, ICL was cut into 28.5 mm×10 cmpieces for a 2 layer graft with approximately a 20% overlap. To supportthe ICL in the formation of the tubes, a cylindrical stainless steelmandrel with a diameter of about 4 mm was covered with KRATON®, anelastic sleeve material that facilitates the removal of the formedcollagen tube from the mandrel and does not adhere or react with theICL.

The flagging apparatus of the invention was used to contact and adherethe edge of a sheet of ICL to a mandrel. The long edge of the ICL wasmoistened with sterile water on the sleeve around the mandrel andadhered to the mandrel and allowed to dry for about 15 minutes to form a“flag”.

The rolling machine of the invention was used to roll a flagged sheet ofICL around the mandrel to form a tube of ICL. The ICL was rolled aroundthe mandrel and over itself one complete revolution. After rolling wascomplete, air bubbles, folds, and creases were smoothed out from underthe material and between the layers. The mandrels and rolled constructswere allowed to sit in the oncoming airflow of the laminar flow cabinetand allowed to dry for about an hour in the cabinet at room temperature,approximately 20° C.

Chemical crosslinking solution of either crosslinked 1 mM EDC or 10 mMEDC/25% acetone v/v in water, in volumes of about 50 mL crosslinkingsolution per tube, was prepared immediately before crosslinking. Thehydrated ICL tubes were then transferred to either of two cylindricalvessels containing either crosslinking agent. The vessel was covered andallowed to sit for about 18±2 hours in a fume hood, after which time thecrosslinking solution was decanted and disposed. ICL tubes were thenrinsed three times with sterile water for about 5 minutes per rinse.

The crosslinked ICL tubes were then removed from the mandrel by pullingthe Kraton sleeve off the mandrel from one end. Once removed, the ICLtubes containing the Kraton were allowed to dry for an hour in a laminarair flow hood. Once dried, the sleeve was removed from the lumen of eachICL tube by pulling it out from one end.

ICL tubes were sterilized in 0.1% peracetic acid at approximately pH 7.0overnight according to the methods described in commonly owned U.S. Pat.No. 5,460,962, the disclosure of which is incorporated herein in itsentirety. The ICL tubes were then rinsed of sterilization solution threetimes with sterile water for about 5 minutes per rinse. The peraceticacid sterilized ICL collagen tubes were then dried in a laminar flowhood and then packaged in sterile 15 mL conical tubes untilimplantation.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious to one of skill in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

1. A method for forming a tube construct from a planar sheet matrix,comprising: flagging a planar sheet matrix by aligning a mandrel alongone edge of the sheet and contacting it to the sheet so that the planarsheet matrix and the mandrel adhere, rolling the flagged planar sheetmatrix around the mandrel while maintaining even tension on the sheetand smoothing out bubbles or creases using a roller as it is rolled ontothe mandrel until the sheet contacts and overlaps itself to a degree toform a bonding region that keeps the tissue in a tubular form.